A fatty acid glyceride is an ester of glycerol and one, two, or three fatty acids. If only one OH group of the glycerol is esterified with a fatty acid, the ester is known as a monoglyceride. If two OH groups of the glycerol are each esterified with a fatty acid, the ester is known as a diglyceride. If all three OH groups of the glycerol are each esterified with a fatty acid, the ester is known as a triglyceride.
In the context of the present invention, the initials “PUFA” stand for “polyunsaturated fatty acid”, i.e. a fatty acid which is at least tri-unsaturated.
An omega-3-fatty acid is an at least tri-unsaturated fatty acid and is thus a PUFA. An omega-3-fatty acid has a double bond between the third and fourth carbon atoms counting from the methyl end, the methyl C atom being counted as the first C atom. Special omega-3-fatty acids are EPA ((all-Z)-5,8,11,14,17-eicosapentaenoic acid) and DHA ((all-Z)-4,7,10,13,16,19-docosahexaenoic acid).
In the prior art, PUFA glycerides, i.e. glycerides in which PUFA acyl groups make up a large proportion of all the fatty acyl groups present, are produced in particular by one of the following two processes:                (1) transesterification of fish oil to ethyl esters, enrichment of the PUFAs by distillation and re-synthesis to glycerides. The triglyceride synthesis is generally carried out enzymatically.        (2) selective hydrolysis to fish oils to enrich the PUFAs in the glycerides and purification of the PUFA glyceride by distillation. The selective hydrolysis is generally carried out enzymatically.        
Process (1), the enzymatic triglyceride synthesis, is described, for example, in EP-A 0 528 844.
Process (2) or the selectivity of lipases for PUFAs in the hydrolysis of glycerides is disclosed in several patent applications, cf. for example WO 97/19601, WO 95/24459, WO 96/37586, WO 96/37587, EP-A 0 741 183, WO 96/26287, WO 00/73254, WO 04/043894, WO 00/49117 and WO 91/16443.
The following is known from the prior art on enzyme selectivities for PUFAs. Most lipases and phospholipases have a negative selectivity for PUFAs by comparison with other fatty acids typically present in vegetable and fish oils. By “negative selectivity” is meant that the lipases hydrolytically split off the other fatty acid groups from glycerides more rapidly than the PUFA acyl groups. Accordingly, the enzymatic enrichment of PUFAs generally proceeds via a modification of the other “non-PUFA” fatty acids. This can be done by esterification, transesterification, or hydrolysis of esters.
Negative selectivities for PUFAs are described, for example, for Candida and Mucor lipases. Some enzymes, for example, those isolated from cold water fish, have a positive selectivity for PUFAs.
The microfungus Thermomyces lanugenosus was formerly known as Humicola lanuginosa. For this reason, there are a large number of scientific treatises on lipase from Thermomyces under the name of Humicola. The lipase from Thermomyces is distinguished in particular by an activity optimum in the alkaline pH range and by high stability, even at a pH of 12 to 13. The enzyme is used in detergents. The articles Biotechnology and Bioengineering 48 (1), 1995; pages 78-88 and Biotechnology and Bioengineering 48 (3), 1995; pages 190-196 provide a good overview of the properties of Thermomyces lipase.
Lipase from Thermomyces lanugenosus is commercially obtainable, for example, as a liquid preparation with the name Lipozym® TL 100 L or Lipolase® 100 L, from Novozymes A/S, Bagsvaerd, Denmark.
The other lipases used in the following Examples are also commercially obtainable. The lipase from Geotrichum candidum was self-produced.
In the following, U stands for “unit” and is an indication of the activity of enzymes. 1 U is the reaction of 1 μmol substance per minute under certain, defined reaction conditions.
Determining the activity of lipase from Thermomyces lanugenosus is carried out as follows (for Novozymes A/S by the method for Lipozym® and Lipolase®): the release of butyric acid from glycerol tributyrate is determined at 30° C./pH 7. A 0.16 M tributyrin solution is used for this purpose and butyric acid is titrated with NaOH at a constant pH. 1 unit corresponds to the activity which releases 1 μmol butyric acid from tributyrin per minute.
Fish oils consist essentially of triglycerides containing a mixture of saturated, mono- and poly-unsaturated fatty acids, more particularly with a high proportion of 5×- and 6×-unsaturated fatty acids, which may be used as a health-promoting food supplement. Since the highly unsaturated fatty acids in particular are health-promoting, there is an advantage in enriching them. This can be done, for example, by selective removal of the non-highly unsaturated fatty acids from the triglycerides, for example through selective enzymatic hydrolysis with lipases.
TABLEtypical fatty acid compositions of various oilsTypical composition of the main constituents of food fats(1 = dairy fat; 2 = porcine tallow, 3 = bovine tallow,4 = sunflower oil, 5 = soybean oil, 6 = olive oil,7 = rapeseed oil, 8 = palm oil, 9 = sardine oil,10 = tuna oil, 11 = hydrogenatedvegetable oil (sunflower) are described as follows.Fatty acid1234567891011C4:03C6:0-C10:05C12:03C14:010341173C16:0332430410114391821<10C16:1433106C18:098224421536>90C18:126463823227559451519C18:2283645392282C18:318101C20:5177C22:6924
Owing to their large number of double bonds, PUFAs are highly temperature- and oxidation-sensitive. Isomers can easily be formed through migration of the double bonds while peroxides and polymers can easily be formed through oxidation. Where polyunsaturated fatty acids are used in food supplements and pharmaceutical products, it is important to avoid such secondary products in the production process. Particularly in the production of highly enriched PUFAs from fish oils, which are particularly interesting, for example, for administration in the form of capsules, a loss of quality of the end product rapidly occurs through thermal enrichment processes.
A typical working-up process known from the prior art is the distillation-based fractionation of, generally, PUFA ethyl esters which are obtained from the fish oils by chemical transesterification. In this process, the PUFA esters are exposed to severe heat stress by—partly—repeated fractionation.
An alternative process is urea fractionation. Although this process does not involve significant heat stress, it is attended by a serious danger in the formation of toxic secondary products which contaminate the PUFAs.
An alternative method to the enrichment of PUFAs is low-temperature enzymatic fractionation. For example, PUFAs can be enriched in the form of their glycerides or their ethyl esters. The enrichment can be carried out by selective hydrolysis, selective synthesis, or selective transesterification.
However, after the selective enzymatic reaction, there remains the problem of separating the various fractions (for example fatty acid and glycerides) which, generally, are completely soluble in one another. The reaction mixtures from the enzymatic enrichment are generally separated by distillation in a second step. This separation also exposes the PUFAs to heat stress with the risk of a loss of quality.
European patent application 06023997 discloses a single-stage process for the production of zinc ricinoleate in which castor oil is hydrolyzed in the presence of suitable enzymes, preferably lipases, and at the same time reacted with zinc oxide in aqueous solution to form zinc ricinoleate.
WO 2005/007864 discloses a process for the production of carboxylic acid salts and, more particularly, a process for the enzymatic hydrolysis of fish oils in alkaline medium in the presence of calcium salts, the calcium salts of the PUFAs present in the fish oils being obtained.
WO 2005/00784 discloses that the most suitable enzyme for the alkaline hydrolysis of esters is lipase from Thermomyces lanugenosus. This lipase was specially developed for detergent applications and shows high activity at alkaline pH values. The lipase is capable of producing soaps (i.e. metal salts) from esters in a one-step reaction in the presence of, for example, Ca(OH)2 or Mg(OH)2. The soaps can then be isolated from the reaction solution by filtration.
WO 07/119811 discloses a method for production of a condensed PUFA oil, which comprises performing an alcoholysis reaction of a PUFA-containing oil-and-fat with a lipase in the presence of at least one compound selected from magnesium oxide, magnesium hydroxide, calcium oxide and calcium hydroxide and a small quantity of water and separating a glyceride fraction.
In principle, most enzymes are capable of catalyzing an ester hydrolysis under alkaline conditions, at least for a short time. Thermomyces lipase has the best price/performance ratio of all commercially obtainable lipases.
By way of the present invention, it has now also been discovered that Thermomyces lipase has a negative selectivity for PUFAs.
The use of alkaline hydrolysis for enriching PUFAs is also not described in the prior art.